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`John Marquis; Jian Wang; Kevin Maresca; Shawn Hillier; Craig Zimmerman; John Joyal; John Babich
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`Author & Article Information
`Cancer Res (2009) 69 (9_Supplement): 4467.
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`Abstract
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`Seprase, also know as fibroblast activating protein alpha (FAP\#945;) is a key component of the
`tumor microenvironment. Its expression is normally restricted to fetal mesenchymal tissue and sites
`of wound healing, but is selectively over expressed in the tumor stroma. It is expressed in cancer-
`associated fibroblasts of greater than 90% of human primary epithelial tumors including breast, lung,
`colorectal, gastric, cervical and ovarian cancers, making it an attractive target to exploit for
`noninvasive radioimaging, as well as targeted radiotherapy of cancer. Seprase is an 88 kDa, type II,
`intregral membrane peptidase in the dipeptidyl peptidase-4 (DPP-IV) family of prolyl peptidases. We
`designed, synthesized and evaluated a series of iodine substituted benzamido-glycine-
`boronoproline analogs as seprase antagonists, with the potential to be radiolabeled with either 123I or
`131I for radiodiagnostic or radiotherapeutic use, respectively. Iodine was substituted at the three
`positions of the benzene ring, and compounds were assessed for their ability to inhibit the enzymatic
`activity of recombinant human seprase in a fluorescence based assay. Among the most active
`compounds, an ortho-iodine analog (MIP-1231) displayed an IC 50 of 6 nM, whereas even more
`potent para- (MIP-1232) and meta-substituted (MIP-1233) analogs both had IC 50 values of 0.6 nM.
`To examine the selectivity for seprase over other prolyl peptidases, compounds were tested for their
`ability to inhibit the enzymatic activity of prolyl oligopeptidase (POP), the only other endopeptidase
`in this family of peptidases. The IC50 values of MIP-1231, MIP-1232, and MIP-1233 for POP were
`58, 19, and 7 nM, respectively, with POP/FAP ratios of 10, 32, and 12, respectively. These data
`demonstrate that although the para- and meta- substituted compounds have similar ability to inhibit
`seprase activity, the para- substituted analog displayed better selectivity. To examine binding to
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`seprase in vivo, human embryonic kidney (HEK-293) cells were stably transfected with the human
`seprase gene, and highly expressing clones were selected and verified by Western blotting. MIP-
`1232 was radiolabeled with 123I for saturation binding analysis using the stable seprase expressing
`
`Petitioner GE Healthcare – Ex. 1035, p. 1
`
`ARTICLE NAVIGATION
`CANCER CHEM ISTRY | MAY 0 1 2009
`Abstract #4467: Targeting tumor microenvironment with
`radiolabeled inhibitors of seprase (FAP\#945;)
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`cells. The Kd of MIP-1232 for seprase was determined to be 30 nM, whereas there was no specific
`binding to a non-expressing clone. The Bmax of the seprase expressing cells was determined to be
`approximately 8 pmol/106 cells. In addition, MIP-1232 was shown to inhibit the seprase enzymatic
`activity of the stable seprase expressing cells. These radiolabeled seprase inhibitors are currently
`being evaluated for tumor uptake and tissue distribution in mice bearing seprase expressing HEK-
`293 xenografts, as well as tumors that promote seprase expression in the tumor associated stroma.
`Radiolabeled seprase inhibitors could be exploited for the diagnosis, staging, prognosis, and
`potential treatment of solid tumors.
`
`Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia
`(PA): AACR; 2009. Abstract nr 4467.
`
`100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO
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`American Association for Cancer Research
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